Project Summary While it is well established that organelles communicate extensively through vesicular transport, recent work has revealed that essential cellular processes including lipid and calcium homeostasis can be coordinated through evolutionary conserved membrane contact sites (MCS). MCS are maintained by tethering proteins which create specialized microdomains that allow the targeted exchange of ions and lipids between the two membranes. Loss of human orthologs of these proteins have been implicated in a broad range of neurodegenerative diseases. However, the molecular function of these proteins, as well as the mechanisms underlying how such sites of inter- organelle contact regulate cellular physiology and pathophysiology remain to be determined. As an ideal entry- point into this question, we have chosen to study the metazoan ortholog of yeast Ist2, the first established membrane contacts site tether, which encodes a TMEM16K transmembrane protein. Loss of TMEM16K in humans is causative for progressive autosomal recessive spinocerebellar ataxia (SCAR10). We have established that a TMEM16K mouse knockout model recapitulates critical aspects of human disease and have found TMEM16K absence leads to perturbed dendritic morphology in Drosophila neurons. We have further established that endoplasmic reticulum (ER) localized TMEM16K directly binds endolysosomal specific phosphatidylinositols and is required for endolysosomal maturation, suggesting that TMEM16K acts as a critical mediator of ER-endolysosomal inter-organelle communication. Endolysosomal maturation is required for proper sorting and degradation of macromolecules, and is emerging as central player in a host of neurodegenerative diseases. The goal of this proposal is to elucidate the conserved molecular mechanisms through which TMEM16K mediates endolysosomal maturation at ER-endolysosomal membrane contact sites, in order to understand their role in neurodegeneration. To accomplish this, we propose to: (1) Identify the interactome of TMEM16K to reveal molecular pathways perturbed in the disease using novel approach based on proximity biotinylation and mass spectrometry; and (2) Functionally analyze how TMEM16K pathway facilitates endolysosomal maturation in health and disease using an in vivo aged Drosophila neuronal model. Overall, by combining novel technical advancements with comparative biology approaches the implementation of this proposal will define the molecular mechanism of how ER-localized TMEM16K facilitates endolysosomal maturation and contributes to the pathophysiology of neurodegeneration. Considering that such communication at membrane contact sites is emerging as a critical regulatory mechanism for cellular homeostasis, with an increasing number of components linked to neurodegenerative disease, their better understanding could also open new avenues for novel therapeutics. .